Insulin (INS) resistance and hypertension often coexist, and frequently progress to diabetes and cardiovascular disease. INS and its structurally and functionally similar peptide, insulin-like growth factor (IGF-1), both have been shown to promote vasorelaxation, as well as glucose (GLU) disposal. Recent studies in our laboratory suggest that these actions of INS/ IGF-1 are mediated through the activation of phosphatidylinositol- 3-kinase (PI3-K) and the downstream, protein kinase B (Akt) signaling cascade. This signaling pathway increases: nitric oxide (NO) production by endothelial cells (EC), vascular smooth muscle cell (VSMC)- sodium pump (Na +, K+-ATPase) and myosin bound phosphatase (MBP) activity and GLU transport in skeletal muscle and adipocytes. Thus, functional alterations in these INS/ IGF-1 signaling pathways are likely to play an important role in these pathologies. In this regard, there is emerging evidence that angiotensin II (Ang II) interferes with this signaling, resulting in a state of resistance to INS/ IGF-1 mediated vasorelaxation and GLU transport; however, the role of intermediary signaling molecules are unclear. We hypothesize that Ang II exerts antagonistic effects on INS / IGF-l-stimulated PI3-K/Akt signaling through the generation of reactive oxygen species (ROS) and activation of RhoA. Further, it is proposed that in states of lNS/ IGF-1 resistance and hypertension there is exaggerated tissue Ang II mediated generation of ROS, and activation of RhoA. To test the main hypothesis and its corollary, we will address the following 2 specific aims: 1. To determine the role of RhoA and ROS in mediating the inhibitory effects of Ang II on the Akt-mediated actions of INS and IGF-1 involved in NO metabolism in EC; VSMC Na+,K+-ATPase; MBP activation; and also in the potentiation of GLU transport in skeletal muscle and adipose tissue. 2. To ascertain the role or Ang H-mediated RhoA/ROS stimulation in the impairment of GLU disposal and vasorelaxation in INS-resistant and Ang II overexpressing rodent models of hypertension. Using primary cultures of EC and VSMC, we will examine the role of Ang II-stimulated RhoA and ROS on NO metabolism in EC and VSMC Na +, K+-ATPase and MBP activity. To delineate the role of Akt, RhoA, and ROS signaling in mediating the counterregulatory actions of Ang II and INS/ IGF-1, we will interrupt each of these signaling pathways by transfecting VSMC with dominant negative constructs of Akt and RhoA, and antisense expression of p22phox, one of the critical components of NAD(P)H oxidase. We will profile and quantify site-specific phosphorylation and ROS-induced posttranslational modifications of the signaling molecules (i.e., Akt, RhoA, phoxes) and enzymes (i.e., eNOS, sodium pump, MBP) using liquid chromatography-tandem mass spectrometry. These studies will be complemented with in vivo and ex vivo studies of vasculature and skeletal muscle/adipocytes in rodent models of hypertension and INS resistance as well as normotensive controls. This proposed investigation should significantly enhance our knowledge regarding the role of Ang II in the pathogenesis of INS resistance and hypertension